Dear Firdous, As Matt refers BeF3 mimics the GTP/ATP bound states. For the transition state we have used mixtures of AlCl3 and HKF2 in combination with GDP (or ADP) and MgCl2 within the buffer. Be aware that depending on the concentrations used of each component you get different proportions of AlF3 and AlF4 (Goldstein G. 1964. Equilibrium distribution of Metal-Fluoride complexes. Analytical Chemistry 36:243–244. DOI: https://doi.org/10.1021/ac60207a074). Also, if you are thinking on doing some biochemistry (binding affinity or similar), you must know about Al3+ contamination in glass material and nucleotide solutions (Sternweis PC, Gilman AG. 1982. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. PNAS 79:4888–4891. DOI: https://doi.org/10.1073/pnas.79.16.4888).
Best, Marian > El 4 ene 2024, a las 11:19, Matthew BOWLER <mbow...@embl.fr> escribió: > > Dear Firdous, > > beryllium fluoride is actually a ground state analogue of GTP as > trifluoroberyllate is tetrahedral. To get a transition sate analogue you need > either AlF4- or MgF3-. Preparation of these complexes is very easy. The great > advantage of metal fluoride transition state analogue and ground state > analogue complexes is the fact that all components are present in solution > and readily self-assemble in the active site forming stable enzyme complexes > that are relevant to the catalytic cycle. The inorganic metal fluoride salts > (AlF3, and MgF2) are too insoluble to use; therefore, the fluoride anion and > metal cation components must be added from separate stock solutions. Both > ammonium fluoride and sodium fluoride are suitable as the source of fluoride > and are readily soluble in water. Metal chlorides can be easily dissolved in > water at high concentration (0.5 M) and the solutions conserved at -20°C. One > of the critical aspects in preparing metal fluoride enzyme complexes is the > pH of the resulting solution. In particular, solutions of AlCl3 and BeCl2 are > highly acidic (pH 2) samples should be prepared in 100 mM unbuffered Tris > base. The optimized sequence of component addition is to add fluoride to the > prepared buffer first, then the metal chloride stock. > > Hope this helps, Matt > > > > > > > > > > > > On 02/01/2024 18:53, Firdous Tarique wrote: >> Hi >> >> I would appreciate it if someone could share with me a step by step protocol >> for making a stable GDP.BeF3 solution which is often used as a transition >> state analogue for structural studies of a protein complex ? >> >> Or a vendor from where it can be purchased directly. >> >> Regards >> >> Firdous >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> > -- > Matthew Bowler > Project Leader MASSIF1@ESRF > European Molecular Biology Laboratory > 71 avenue des Martyrs > CS 90181 F-38042 Grenoble > France > =================================================== > Tel: +33 (0) 4.76.20.76.37 > Fax: +33 (0) 4.76.20.71.99 > > http://www.embl.fr/ > http://www.esrf.eu/MASSIF1 > https://twitter.com/id30_massif1 > https://www.embl.org/people/person/mbowler/ > =================================================== > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
